Transient opening of fibronectin type III (FNIII) domains: the interaction of the third FNIII domain of FN with anastellin.
Transient opening of fibronectin type III (FNIII) domains: the interaction of the third FNIII domain of FN with anastellin.
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DOI：
10.1021/bi900001g
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发表时间：
2009-05-19
期刊：
影响因子：
2.9
通讯作者：
Erickson, Harold P.
中科院分区：
文献类型：
--
作者：
Ohashi, Tomoo;Augustus, Anne Marie;Erickson, Harold P.
我们之前报道过，FN 的纤连蛋白 (FN) III 型结构域可能展开与 anastellin 相互作用并形成 FN 聚集体。在本研究中，我们重点关注阿那斯特林和第三个 FN III 型结构域 (III3) 之间的相互作用，该结构域是 FN 上关键的阿那斯特林结合位点。通过 8-苯胺基-1-萘磺酸盐 (ANS) 荧光监测 Anastellin 与 III3 的结合。 ANS 与 anastellin 的结合显着增加了其发射强度，但通过添加 III3，发射强度降低了一半，表明 ANS 和 III3 在 anastellin 上共享一个共同的疏水结合位点。通过链内二硫键稳定的 III3 工程突变体不会与 anastellin 相互作用，从其未能干扰 ANS 与 anastellin 的结合即可看出。我们还突变了疏水性核心残基以破坏 III3 的稳定性，并发现这些突变体仍然能够与 anastellin 相互作用。 Anastellin 与 III3 的结合也使用基于 GFP 的分子内 FRET 构建体进行监测，其中 III3 两侧有两个 GFP 变体 (III3-FRET)。 Anastellin 与 III3-FRET 结合并导致 FRET 信号增加。解离常数估计为~210 nM。 anastellin 与 III3-FRET 的结合动力学符合一级反应，半衰期约为 30 s；不稳定的 III3 突变体的动力学甚至更快。 MALDI-TOF 质谱表明 III3 的中间部分在安那斯林结合后变得不稳定且对蛋白酶敏感。因此，III3 的稳定性似乎是 anastellin 结合的关键因素。
We previously reported that the fibronectin (FN) type III domains of FN may unfold to interact with anastellin and form FN aggregates. In the present study, we have focused on the interaction between anastellin and the third FN type III domain (III3) which is a key anastellin binding site on FN. Anastellin binding to III3 was monitored by 8-anilino-1-naphthalene sulfonate (ANS) fluorescence. ANS binding to anastellin dramatically increased its emission intensity, but this was reduced to half by the addition of III3, suggesting that ANS and III3 share a common hydrophobic binding site on anastellin. An engineered mutant of III3 that was stabilized by an intrachain disulfide bond did not interact with anastellin, as seen by its failure to interfere with ANS binding to anastellin. We also mutated hydrophobic core residues to destabilize III3 and found that these mutants were still capable of interacting with anastellin. Anastellin binding to III3 was also monitored using an intramolecular GFP-based FRET construct, in which III3 was flanked by two GFP variants (III3-FRET). Anastellin bound to III3-FRET and caused an increase in the FRET signal. The dissociation constant was estimated to be ~210 nM. The binding kinetics of anastellin to III3-FRET fit a first order reaction with a half time of ~30 s; the kinetics with destabilized III3 mutants were even faster. MALDI-TOF mass spectrometry suggested that the middle part of III3 became destabilized and protease sensitive upon anastellin binding. Thus, the stability of III3 seems to be a key factor in anastellin binding.
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